Rita de Cássia Lemos Lima, Fernanda Cristina Ferrari, Maíra Ribeiro de Souza, Bruna Maria de Sá Pereira, Carmen Aparecida de Paula, Dênia Antunes Saúde-Guimarães

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Research Paper

Effects of extracts of leaves from

Sparattosperma leucanthum

on hyperuricemia and gouty arthritis

Rita de Cássia Lemos Lima, Fernanda Cristina Ferrari, Maíra Ribeiro de Souza,

Bruna Maria de Sá Pereira, Carmen Aparecida de Paula, Dênia Antunes Saúde-Guimarães

n Laboratório de Plantas Medicinais, Escola de Farmácia, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, s/n. Ouro Preto,

Minas Gerais CEP 35400-000, Brazil

a r t i c l e

i n f o

Article history: Received 15 August 2014 Received in revised form 25 November 2014 Accepted 29 November 2014 Available online 8 December 2014

Keywords:

Sparattosperma leucanthum Hyperuricemia

Xanthine oxidase Gout

Inﬂammation

a b s t r a c t

Ethnopharmacological relevance: The species Sparattosperma leucanthum (Vell.) K. Schum is used in Brazilian folk medicine to treat rheumatism, throat ulcers, stomatitis, syphilis, bladder stones and as blood cleanser. The aim of this study was to evaluate the potential,in vitroandin vivo, of the extracts of leaves from Sparattosperma leucanthum to treat hyperuricemia and inﬂammation in the gouty arthritis model.

Materials and methods:Ethyl acetate extract (SLE), methanolic extract (SLM) and aqueous extract (SLW) were evaluatedin vitroon XO inhibitory activity andin vivoin an experimental model with oxonate-induced hyperuricemia in mice which was used to evaluate anti-hyperuricemic activity and liver xanthine oxidase (XOD) inhibition. Anti-inﬂammatory activity was also investigated on MSU crystal-induced paw edema model.

Results: Sparattosperma leucanthumcrude extracts showed expressive results on urate-lowering activity in blood. SLW at the dose of 125 mg/kg has proved to be active in reducing hyperuricemia and was capable to inhibit the hepatic xanthine oxidase enzyme (XOD). SLM showed anti-hyperuricemic activity on all doses tested; however, this extract showed activity on the XOD only at the dose of 500 mg/kg. SLE, at the three evaluated doses, has proved to be active in reducing hyperuricemiain vivoand was able to inhibit XO activityin vitroat the concentration of 100mg/mL. This extract was also able to inhibit XOD activityin vivoat the doses of 250 mg/kg and 500 mg/kg. SLE (125 and 250 mg/kg) and SLW (500 mg/kg) showed signiﬁcant anti-inﬂammatory activity on monosodium urate crystal-induced paw edema model.

Conclusions: The ethyl acetate, methanolic and aqueous extracts ofSparattosperma leucanthumshowed signiﬁcant results on evaluated models and therefore may be important agents for the treatment of gouty arthritis and hyperuricemia.

1. Introduction

Hyperuricemia is characterized by an increase in uric acid blood levels due to a metabolic disorder on its production or on its excretion (Haidari et al., 2008). Uric acid is the last metabolite of the purine catabolic pathway in humans. Uric acid is formed by oxidation of hypoxanthine to xanthine and xanthine to uric acid by the enzyme xanthine oxidase (XO). Gouty arthritis is a worldwide disease which is intimately associated to hyperuricemia, whereas this disorder is characterized by precipitation of monosodium urate crystals in the joints leading to an intense inﬂammatory

process and consequently, pain (Schumacher, 2008). The thera-peutic approach against gout is given by the treatment of infl am-mation and the control of hyperuricemia. Nowadays, allopurinol is the only drug used on the control of hyperuricemia that acts by inhibiting hepatic xanthine oxidase enzyme (XOD) activity, then reducing serum uric acid levels. However, allopurinol is not an effective drug to treat acute gout attacks (Dubchak and Falasca, 2010) and its use has been associated with adverse effects, such as fever, skin rashes, allergic reactions, hepatitis and nephropathy (Umamaheswari et al., 2009). Nonsteroidal anti-inflammatory drugs such as indomethacin are frequently used in thefirst-line

therapies for acute inﬂammation in gout; however these drug may develop serious side effects such as gastrointestinal toxicity, renal toxicity or gastrointestinal bleeding (Cronstein and Terkeltaub, 2006). Therefore, the search for new drugs with anti-inﬂ am-matory and urate-lowering activities, including xanthine oxidase Contents lists available atScienceDirect

journal homepage:www.elsevier.com/locate/jep

Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.11.051

n

Corresponding author. Tel.:þ55 31 3559 1031. E-mail addresses:saude@ef.ufop.br,

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inhibitors, has been the focus of several studies (Zhu et al., 2004; Mo et al., 2007; Ahmad et al., 2008; Haidari et al., 2008; Liu et al., 2008; Huang et al., 2011; Sabina et al., 2011; De Souza et al., 2012). Sparattosperma leucanthum(Vell.) K. Schum, popularly known as “cinco-chagas”, “cinco-folhas” or “caroba-branca” (Correa, 1926–

1978), is a species from Bignoniaceae family largely distributed in Atlantic forest, Amazon forest and Cerrado regions in Brazil (Lorenzi, 2002). Ethnobotanical studies relate the use of Sparattos-perma leucanthum in Brazilian folk medicine as anti-rheumatic, anti-inflammatory, blood cleanser, in the treatment of thr-oat ulcers, stomatitis, syphilis, skin rashes and bladder stones (Correa, 1926–1978; Patzlaff, 2007; Leite et al., 2008; Crepaldi and Peixoto, 2009; Costa and Leitão, 2011; Oliveira et al., 2011). Previous phytochemical studies aboutSparattosperma leucanthum described the isolation of theflavone pinocembrin-7-O-(-d-neo-hesperidoside), a derivative of the flavone pinocembrin, which

presents signiﬁcant antioxidant activities (Costa and Leitão, 2011; Rasul et al., 2013). Recent studies have shown the relation between antioxidant and anti-inﬂammatory activities, which is given by

inhibiting the action of ROS, NO and other free radicals that are involved with the inﬂammatory process, particularly on acute gout attacks (Busso and So, 2010). Therefore, the aim of this study was to evaluate,in vitro and in vivo, the xanthine oxidase inhibitory activity and the anti-hyperuricemic and anti-inﬂammatory activ-itiesin vivoofSparattosperma leucanthumcrude extracts.

2. Material and methods

2.1. Chemicals

Xanthine oxidase (EC 1.1.3.22) from cow's milk (1.3 units/mL), xanthine, potassium oxonate, allopurinol and indomethacin were purchased from Sigma-Aldrich. Uric acid assay kit was purchased from Bioclin (Minas Gerais – Brazil). Monosodium urate (MSU) crystals were prepared according to the previously described method (Rasool and Varalakshmi, 2006; Sabina et al., 2011). Methanol, ethyl acetate, hexane, dimethilsulfoxide (DMSO) and Tween 80 were of analytical grade.

2.2. Plant material

Leaves fromSparattosperma leucanthum(Vell.) K. Schum were collected in Minas Gerais, Brazil, in March, 2011, with permission of Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio/Sistema de Autorização e Informação em Biodiversidade– SISBIO (license no. 17021-5). A voucher specimen (OUPR 28217) is deposited at the herbarium of Instituto de Ciências Exatas e Biológicas–UFOP, Ouro Preto, Brazil.

2.3. Preparation of plant extracts

The leaves were dried in an air circulating oven at 371C for two weeks, and pulverized. Part of the obtained powder (1.2 kg) was exhaustively extracted with each solvent used, by percolation, at room temperature. The plant material was ﬁrst extracted with hexane until exhaustion of the vegetable material with this solvent. After, it was exhaustively extracted with ethyl acetate and ﬁnally with methanol. The solvents were removed under reduced pressure, at 401C, in order to obtain dried crude hexanic extract (SLH, 40.8 g), 130.6 g of dried crude ethyl acetate extract (SLE) and dried crude methanolic extract (SLM, 90.0 g). The aqueous extract was obtained by exhaustive extraction of 72.0 g of powder plant material with distillated water, by percolation, and then, the water was removed by lyophilization, yielding 12.1 g of dried crude aqueous extract (SLW).

2.4. Physical and chemical color reaction and TLC analysis

The physical and chemical color reactions were performed with the extracts as previously described (Farnsworth, 1966; Marini-Betollo et al., 1981; Wagner et al., 1984; Matos, 1997). The

flavonoids were identified by hydrochloric acid–magnesium reac-tion and TLC sprayed with NP/PEG reagent. Anthocyanins and catechins were assayed using hydrochloric acid and sodium hydroxide. Tannins were analyzed using ferric trichloride and potassium ferrocyanide. The presence of saponins was evaluated using froth test and the Lieberman–Burchard reaction. Coumarins, quinones and anthraquinones were identified by alkali reaction. The presence of triterpenes and steroids were assayed using the Lieberman–Burchard reaction. Alcaloids were analyzed by the Dragendorff reaction. Analyzes were carried out using thin layer chromatography and colorimetric pharmacognostic tests.

2.5. Evaluation of in vitro xanthine oxidase (XO) inhibition activity of SLE, SLM and SLW

The assay with crude extracts of Sparattosperma leucanthum was carried out using a previous methodology described by Ferraz-Filha et al. (2006), with modiﬁcations. For extracts with an inhibitory activity of more than 25% at 100mg/mL, the IC50 values were determined. These assays were realized with extract concentrations from 10 at 100mg/mL, in triplicate. Thus, inhibition percentages are the mean of triplicate observations.

Initially, 6.4 mg of crude extracts (SLE, SLM and SLW) was solubilized in 100mL of DMSO and then diluted with water, yielding a 640mg/mL solution of each extract. Thereafter, 500mL of each solution was added in 1.3 mL of phosphate buffer 65 mM (pH 7.4) containing 0.28 U/mL of XO and incubated for 10 min at 301C in order to promote the interaction between the compounds of the extracts and XO enzyme. After the incubation period, 1.4 mL of substrate xanthine (0.6 mM) was added into the test solution. The absorbance of each sample was measured, spectrophotome-trically (Varian BIO-50), at every minute for 10 min in a wave-length of 295 nm. Allopurinol (positive control) was tested in the same conditions than the crude extracts, at aﬁnal concentration of

10mg/mL. XO inhibitory activity was expressed as the percentage

inhibition of XO in the above assay mixture system, calculated as: % inhibition¼(1 test inclination/blank inclination)100.

2.6. Animals

Male albino Swiss mice (25–30 g) were supplied by Universi-dade Federal de Ouro Preto. Animals were divided into experi-mental groups (n¼6), housed in plastic cages and maintained on a 12-h light/12-h dark cycle. They were given standard chow and waterad libitum. All experimental procedures were approved by the Ethical Committee of Universidade Federal de Ouro Preto, Brazil (no. 2012/65).

2.7. Preparation of drugs and test solutions for in vivo methods

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2.8. Anti-hyperuricemic effects in oxonate-induced hyperuricemic mice and inhibition of liver XOD activity

In order to evaluate the anti-hyperuricemic activity of Spar-attosperma leucanthumextracts, an experimental model of hyper-uricemia induced by potassium oxonate was adopted according to De Souza et al. (2012), with modifications. Animals were divided into 12 experimental groups (n¼6) and fasted 2 h before drug administration. Potassium oxonate, an uricase inhibitor, was administrated to the animals of groups 2–12 (250 mg/kg, I.P.) in thefirst and third days of the experiment. Oral treatments were given by gavage, 1 h after the potassium oxonate administration, once a day, for 3 days of experiment. Experimental group 1 (normal control group) and group 2 (hyperuricemic control group) received only vehicle by oral route. Group 3 was treated with allopurinol (10 mg/kg, P.O.). Experimental groups 4–12 were treated with SLE, SLM and SLW, at the doses of 125, 250 and 500 mg/kg, once a day, for 3 experimental days. At the third day, animals were anesthetized with an association of ketamine and xylasine (100 and 20 mg/kg, respectively), intraperitoneally, 1 h afterfinal drug administration, in order to collect the blood from abdominal aorta. Blood samples were maintained at room tem-perature until blood coagulation and then centrifuged at 1500gfor 5 min. Supernatant were collected and centrifuged at 3000g for 10 min until complete sera separation. These materials were stored at 201C until uric acid quantification. Mice livers were also collected, washed in 0.9% cold saline solution and stored at

801C until XOD activity determination.

2.8.1. Uric acid assay

An enzymatic-colorimetric method, using a standard diagnostic kit (Bioclin, Brazil), according to manufacturer's instructions, was used in order to evaluate serum uric acid concentration.

2.8.2. Assays inhibition of liver XOD activity

The residual activity of XOD was spectrophotometrically deter-mined monitoring uric acid formation from xanthine, as pre-viously described byDe Souza et al. (2012), with modiﬁcations. Initially, mice livers were homogenized in 5 mL of 80 mM sodium phosphate buffer (pH 7.4). The homogenates were centrifuged at 3000gfor 10 min at 41C. Then, the lipidic layer was removed and the supernatant was centrifuged at 10,000gfor 60 min at 41C. The supernatant obtained was used in the XOD residual activity test and protein dosing. In test tubes containing 100mL of liver homogenate and 5.4 mL of potassium oxonate solution (1 mM) in50 mM sodium phosphate buffer (pH 7.4) previously incubated at 351C for 15 min was added 1.2 mL of xanthine solution (250 mM). The reaction was stopped after 0 and 30 min by adding 0.15 mL of 0.6 M HCl. Thereafter, test tubes were centrifuged at 3000gfor 5 min and the absorbance of supernatant was measured at 295 nm using a Varian 50Bio UV/VIS spectrophotometer. Total protein concentration was determined spectrophotometrically according to Bradford (1976). XOD activity was expressed as nanomoles of uric acid formed per minute per milligram protein.

2.9. Effects of SLE, SLM and SLW of Sparattosperma leucanthum on gouty arthritis inﬂammation model

In order to evaluate the anti-inﬂammatory activity of Sparat-tosperma leucanthum crude extracts on gout, an experimental model, previously described byDe Souza et al. (2012), was carried out with modiﬁcations.

The assay was carried out in a period of three days. The animals were divided into 12 experimental groups (n¼6). On the_ﬁrst day of the experiment, the inﬂammation was induced by intradermal

injection of 0.1 mL (4 mg) of MSU suspension into the mice right hind paw in all animals of groups 2–12. Animals of normal control group (group 1) received 0.1 mL of 0.9% sterile saline solution by intradermal injection into the right hind paw and were orally treated with vehicle, once a day, during the 3 days of experiments. Animals of group 2 were also treated with vehicle and were used as MSU-induced control group. Group 3 (positive control) was orally treated with indomethacin (3 mg/kg). Animals of groups 4–12 were treated by oral route with SLE, SLM and SLW, at the doses of 125, 250 and 500 mg/kg. Treatments were administered on the ﬁrst experimental day, 1 h before MSU injection, and repeated daily for 2 more days.

Paw thickness was measured with a caliper rule (150 mm–6 in., Vonder, China)at 4 h, 24 h, 48 h and 72 h after MSU injection and inﬂammatory swelling was expressed as thickness variation (

Δ

).

2.10. Statistical analysis

In thein vitroevaluation of XO inhibition activity, the IC50was obtained for extracts which presented an activity above 25.0% using GraphPad Prism 5.1 Software (Inc., San Diego, CA, U.S.A.). The IC50values of the extracts were calculated from regression lines of a plot of % inhibition versusextract concentration. These assays were realized with extract concentrations from 10 at 100mg/mL.

Experimental data were analyzed using GraphPad Prism 5.1 Software and results were presented as mean values7S.E.M.. The statistical evaluation of results from hyperuricemic and MSU crystal-induced paw edema assays was analyzed utilizing One-way variance analysis (ANOVA), followed by Newman-Keul's test. Pvaluesr0.05 were considered statistically signi_ﬁcant.

3. Results

3.1. Physical and chemical color reactions and TLC

Preliminary phytochemical analyses are important methodolo-gies for prediction of groups of secondary metabolites present in plants. Therefore, analyses were performed to detect classes of secondary metabolites in extracts ofSparattosperma leucanthum. In SLE were detectedﬂavonoids (aglycone), anthraquinones/qui-nones, triterpenes and steroids. In SLM were identiﬁed tannins,

ﬂavonoids (glycosides and aglycone) and anthraquinones

glyco-sides. In SLW were detected tannins,ﬂavonoids glycosides, anthra-quinones glycosides, coumarins and saponins.

3.2. In vitro xanthine oxidase inhibitory activity

The positive control, allopurinol, a known xanthine oxidase inhibitor, showed an inhibition of 95.63% at the concentration of

10mg/mL and IC50of 0.328mg/mL. The ethyl acetate crude extract

(SLE) showed an inhibition of 46.0% and IC50of 107.0mg/mL. The methanolic (SLM) and aqueous (SLW) crude extracts showed inhibitions of 23.50% and 13.10%, respectively, as shown inTable 1.

Table 1

In vitroinhibitory xanthine oxidase activity of crude extracts ofSparattosperma leucanthum.

Test solution Concentration (mg/mL) Inhibition (%7S.D.) IC50(mg/mL)

Allopurinol 10 95.63 0.328

SLE 100 46.0070.223 107.00

SLM 100 23.5070.248 –

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3.3. Effects of SLE, SLM and SLW on serum urate levels in hyperuricemic mice

Potasium oxonate solution was able to signiﬁcantly increase serum urate levels compared to normal control group (Fig. 1). Allopurinol (10 mg/kg) promoted a signiﬁcant reduction on serum

urate levels of hyperuricemic mice to values lower than that found in normal animals. Treatments with SLE and SLM were able to signiﬁcantly reduce serum urate levels compared to hyperuricemic control group in the three doses evaluated. SLW signiﬁcantly

reduced serum urate levels at 125 mg/kg.

3.4. Effects of SLE, SLM and SLW on XOD activity in mice liver

SLE signiﬁcantly inhibited liver XOD activity at the doses of 250

and 500 mg/kg by 35.10% and 37.33%, respectively (Table 2). SLM was able to inhibit XOD activity at 500 mg/kg by 32.88%. SLW, at 125 mg/kg, inhibited XOD activity by 34.00% when compared to hyperuricemic control group. Allopurinol inhibited XOD activity by 74.16%.

3.5. Effects of SLE, SLM and SLW on monosodium urate crystal-induced inﬂammation in mice

MSU crystals injection induced a significant increase in paw thickness when compared to negative control (Fig. 2). Paw edema was found to be reduced in mice treated with SLE at 250 mg/kg at 4 h, 24 h and 72 h. SLM was not able to reduce significantly paw edema in any of the doses tested. SLW was able to reduce significantly paw edema at 500 mg/kg at 72 h. Indomethacin

(3 mg/kg) has also shown a signiﬁcant anti-inﬂammatory activity.

4. Discussion

Uric acid is the last metabolite of the purine catabolic pathway in humans (Dröge, 2002). In some pathologic conditions, the organism loses the ability to eliminate uric acid and, consequently, an increase in its levels can lead to the precipitation of mono-sodium urate crystals in the joints, which, in turn, causes gouty arthritis or gout (Chiang et al., 1994; Busso and So, 2010).

Allopurinol is one of the main drugs used to treat and control hyperuricemia. This drug presents an effective inhibitory activity of xanthine oxidase, but its use can cause several adverse effects to patients, and these damages are reported as the main cause of abandonment of anti-hyperuricemia therapy (Umamaheswari et al., 2009). As nowadays, none of the clinically available medi-cines can be used to treat both hyperuricemia and the inﬂ amma-tory process that occurs in gouty arthritis (Ahmad et al., 2008) and facing the need to new therapeutics options to treat gout and its consequences, the present study aimed to evaluate anti-hyperuricemic and anti-inﬂammatory activities ofSparattosperma leucanthum leaves extracts, as well as elucidate their possible action pathways in experimental modelsin vivo.

SLW was effective on reducing uric acid levels and inhibiting XOD activity, which indicates that this extract may accomplish its anti-hyperuricemic effect mainly through this action mechanism. SLE was able to reduce uric acid levels at three evaluated doses, and signiﬁ -cantly inhibited liver XOD activity at 250 and 500 mg/kg. Besides showed an inhibition in vitro of XOD activity indicating that this extract can contain substances with different potencial to inhibit the XOD activity. SLM signiﬁcantly reduced serum uric acid levels at all

doses tested; however, it did not show XOD inhibitory activity, which indicates that this extract may accomplish its anti-hyperuricemic effect mainly through other action mechanisms.

The experiment using MSU crystal-induced inflammation has been used to simulate acute attacks of gout occur due to urate crystals accumulation in the joints, which promotes the beginning of a chain of inflammatory reactions such those related to synovial cells activation (especially leukocytes) leading to acute infl amma-tion and promoting tissue damaging (Rasool and Valarakshmi, 2006; Martinon et al., 2006). In MSU crystal-induced infl amma-tion, SLE showed significant results in paw edema reduction at 250 mg/kg, at 4 h, 24 h, 48 h and 72 h. These results can be considered promising, since SLE also presented significant

anti-hyperuricemic and liver XOD inhibitory activities at the same dose in hyperuricemic mice.

The detection of flavonoids, triterpene and steroids in SLE, flavonoids glycosides in SLM andflavonoids glycosides and sapo-nins in SLW indicate that this secondary metabolites may be responsible for anti-hyperuricemic and anti-inflammatory effects

of the extracts, since there are previous reports of such activities for these compounds (Fang et al., 2005; Chen et al., 2006, 2011; Mo et al., 2007; Nguyen and Nguyen, 2013; Nkanwen et al., 2013; Wu et al., 2014). However, additional studies are required to isolate and identify the bioactive substances present in SLE, SLM and SLW.

Fig. 1.Anti-hyperuricemic effects of Sparattosperma leucanthum ethyl acetate extract (SLE), methanolic extract (SLM) and aqueous extract (SLW) in mice pretreated with potassium oxonate. Experiments were performed as described in Section 2. Data present mean7S.E.M. (n¼6). One-way ANOVA followed by New-man-Keul's test was used for statistical signiﬁcance. *Po0.001 compared with hyperuricemic control group;#_P_o_{0.001 compared to normal control group.}

Table 2

Effects ofSparattosperma leucanthumethyl acetate extract (SLE), methanolic extract (SLM) and aqueous extract (SLW) on xanthine oxidase activity in mice liverin vivo.

Treatment Dose (mg/

kg)

XOD activity (U/mg protein)

Inhibition (%)

Control Normal – 15.7071.376 –

Hyperuricemic control

– 16.1870.686 –

Allopurinol 10 4.18070.219nn

74.16

SLE 125 13.4771.883 16.75

250 10.5070.566n _35.10

500 10.1470.638n _37.33

SLM 125 14.9071.937 7.91

250 14.3471.710 11.37

500 10.8670.707n

32.88

SLW 125 10.6871.035n

34.00

250 16.3670.275 –

500 16.0671.532 –

Data represent mean7S.E.M. (n¼6). One-way ANOVA followed by Newman-Keul's test was used for statistical signiﬁcance.

U¼nanomole uric acid/min.

n

Po0.05, compared to hyperuricemic control group.

nn

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5. Conclusion

Sparattosperma leucanthumethyl acetate extract was efﬁcient

in reducing hyperuricemia and MSU crystal-induced infl amma-tion, as well as in inhibiting the in vitro and in vivo xanthine oxidase activity, which indicates that this extract may accomplish its effects mainly through liver XOD inhibition. Aqueous extract, at 125 mg/kg, showed significant anti-hyperuricemic activity and was able to inhibit XOD, which is very important, since in folk medicine Sparattosperma leucanthum is used as tea. Methanol extract was able to reduce serum urate levels in hyperuricemic mice; however its activity could be given by a different pathway, which gives a very important characteristic to Sparattosperma leucanthum. Flavonoids, saponins, triterpene and steroids, detected in extracts, may be the bioactive constituents. Therefore, Sparat-tosperma leucanthumextracts are promising agents for the treat-ment of hyperuricemia and gouty arthritis since they possess both anti-hiperuricemic and anti-inflammatory properties.

Acknowledgments

The authors would like to thank FAPEMIG, REDE TOXIFAR/ FAPEMIG - CBB - RED-00008-14 (Rede Mineira de Ensaios Tox-icológicos e Farmacológicos/ Fundação de Amparo à Pesquisa de Minas Gerais); CAPES (Coordenação de Aperfeiçoamento de Pes-soal de Ensino Superior), CNPq (Conselho Nacional de Pesquisa), Brazil companies, and UFOP (Universidade Federal de Ouro Preto), Brazil forﬁnancial support.

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